Nucleic acid encoding TGF-β and its uses

ABSTRACT

Nucleic acid encoding TGF-β has been isolated and cloned into vectors which are replicated in bacteria and expressed in eukaryotic cells. TGF-β is recovered from transformed cultures for use in known therapeutic modalities. Nucleic acid encoding TGF-β is useful in diagnosis and identification of TGF-β clones.

This application is a continuation application of co-pending applicationSer. No. 07/389,929 filed on 04 Aug. 1989, now U.S. Pat. No. 5,168,051,which is a continuation application of U.S. Ser. No. 07/025,423, filedon 13 Mar. 1987, now U.S. Pat. No. 4,886,747, which is acontinuation-in-part application of U.S. Ser. No. 06/715,142 filed on 22Mar. 1985, now abandoned.

Peptides which can induce the reversible phenotypic transformation ofmammalian cells in culture have been given the name transforming growthfactor (TGF)¹,2. Type α TGF competes with epidermal growth factor (EGF)for binding to the same cell surface receptor³. A 50 amino acid TGF-αspecies has been purified and shown to share sequence homology withEGF⁴. TGF-α is synthesized by various transformed cell lines³,5,6,91.The 50 amino acid TGF-α is initially synthesized as part of a 160 aminoacid precursor molecule which undergoes N- and C-terminal proteolyticprocessing to yield the mature peptide⁷,8. The detection of TGF-αspecies with apparently higher molecular weights¹,2,9 might be due tovariable processing of the 160 amino acid precursor⁹².

Type β TGF activity has been isolated from tumor cells as well as manynormal tissues¹⁰,11, including kidney¹², placenta¹³ and bloodplatelest¹⁴,15. TGF-β is present in platelets, which also containplatelet-derived growth factor (PDGF) and an EGF-like peptide¹⁶. BovineTGF-β has been demonstrated to accelerate wound healing in rats¹⁷ and toinduce fetal RMM cells to undergo differentiation and synthesizecartilage-specific macromolecules⁸⁰. Treatment of NRK fibroblasts withTGF-β does, however, result in an increase in the number of membranereceptors for EGF²⁰. This observation is in agreement with the knownability of TGF-β to greatly potentiate the activity of EGF and TGF-60 onthese cells¹⁰,11. Moreover, TGF-β alone can induce AKR-2B fibroblasts toform colonies in soft agar²¹. In addition to its ability to stimulatecell proliferation, TGF-β has been demonstrated to inhibit theanchorage-dependent growth of a variety of human cancer cell lines¹³. Itis now thought that TGF-β may be identical or very similar to a growthinhibitor isolated from African green monkey (BSC-1) cells²⁴. WhetherTGF-β acts to stimulate or inhibit the growth of a particular cell linetype appears to depend on many variables, including the physiologicalcondition of the cell and the presence of additional growth factors.

Bovine TGF-β has been purified to sequenceable grade (U.S. Ser. No.500,833, filed Jun. 3, 1983) abandoned. The first 15 amino-terminalresidues of the mature protein were found to beAla-Leu-Asp-Thr-Asn-Tyr-CMC-Phe-Ser-Ser-Thr-Gly-Lys-Asn-CMC-, whereinCMC is S-carboxymethyl cysteine representing cysteine or half-cystineresidues.

Human TGF-β from human placenta and platelets has been purified to thesame degree (respectively, U.S. Ser. Nos. 500,927 and 500,832, bothfiled Jun. 3, 1983) and now abandoned. Placental TGF-β was reported tohave the following amino terminal sequence:Ala-Leu-Asp-Thr-Asn-Tyr-CMC-Phe-(Ser-Ser)-Thr-Glu-Lys-Asn-CMC-Val-X-Gln-Leu-Tyr-Ile-Asp-Phe-X-(Lys)-Asp-Leu-Gly-,wherein X was undetermined and CMC is as defined above. Platelet TGF-βwas reported as the amino terminal sequenceAla-Leu-Asp-Thr-Asn-Tyr-X-Phe-Ser-, wherein CMC and X are as definedabove.

Human TGF-β was reported to be composed of two polypeptide chains ofvery similar molecular weight (M_(r) =12,500) which are maintained incovalent association by disulfide bonds. The disulfide bonds wereconsidered likely to play an important role in conferring structure onthe TGF-β molecule (U.S. Ser. No. 500,832).

Several other factors have been described that are related to TGF-β bylimited amino acid sequence homology. The inhibin A and B beta chainsare related to TGF-β by the placement of homologous cysteine residuesand other limited amino acid sequence homology, from which it has beeninferred that inhibin, or more accurately activin (dimers of the inhibinbeta_(A) or beta_(B) chains), is structurally related to TGF-β. Inhibinrepresses the release of FSH from the pituitary, while activin enhancesthe release of FSH⁸¹,82. TGF-β is not known to have this activity.

Mullerian inhibitory substance has a C-terminal region which ishomologous with TGF-β and inhibits the growth of Mullerian-derivedtumors⁸³,84.

TGF-β prepared by purification from biological materials presents a riskof contamination by infectious agents such as HTLV-III or hepatitisviruses. Accordingly, it is an object of this invention to prepare TGF-βfrom sources that do not present a risk of contamination.

It is another object to prepare nucleic acid that will hybridize withDNA encoding biologically active TGF-β. When appropriately labelled,this nucleic acid is useful in diagnostic assays for TGF-β mRNA and inisolating DNA encoding TGF-β.

It is further object herein to prepare vectors containing DNA thatencodes TGF-β, together with host cell transformants that expressbiologically active TGF-β.

SUMMARY

In accordance with this invention, the foregoing objects are achieved bya method comprising (a) constructing a vector which includes nucleicacid encoding TGF-β, (b) transforming a heterologous host eukaryoticcell with the vector, (c) culturing the transformed cell, and (d)recovering TGF-β from the culture.

Nucleic acid encoding two subtypes of TGF-β (TGF-β₁ and TGF-β₃) isprovided which is useful in constructing the above vectors. This nucleicacid or a nucleic acid capable of hybridizing therewith also is labelledand used in diagnostic assays for DNA or mRNA encoding TGF-β or relatedproteins.

The preparation of TGF-β derivatives by recombinant methods is madepossible by knowledge of the TGF-β coding sequences disclosed herein.These derivatives include silent and expressed mutants in the nucleicacid encoding TGF-β.

Silent variants involve the substitution of one degenerate codon foranother where both codons code for the same amino acid, but whichsubstitution could exert a salutary effect on TGF-β yield in recombinantculture, e.g. by modifying the secondary structure of TGF-β mRNA, andwhich salutary substitution is identified by screening TGF-β yields fromtransformants.

Expressed TGF-β variants fall into one or more of three classes:deletions, substitutions or insertions. Deletions are characterized bythe elimination of amino acid residues without the insertion of areplacement residue. Deletional variants of TGF-β are useful in makingTGF-β fragments, for example, where it is desired to delete an immuneepitope.

Substitution variants are those in which one amino acid residue has beenreplaced by another. Such variants are extremely difficult to make bymethods other than recombinant synthesis, especially substitutionstargeted for the interior of the primary amino acid sequence. They areuseful in modifying the biological activity of TGF-β. Substitutionvariants include allelic forms of TGF-β as well as TGF-β subtypes.

TGF-β is found as a disulfide linked dimer. Further variants includeheterodimers of TGF-β subtypes and heterodimers of one TGF-β chainhaving a native amino acid sequence disulfide bonded through theordinary homodimer disulfide linkages to a predetermined variant ofTGF-β. In this case, such variants include biologically active as wellas biologically inactive TGF-β.

Insertional variants are those in which one or more residues are placedwithin the internal TGF-β sequence or at either end thereof. Variants ofthis class include fusion proteins resulting from insertions at thecarboxyl or amino terminal residues of TGF-β. TGF-β fusions withbacterial or other immunogenic proteins are useful for raisingantibodies against TGF-β or its predetermined fragments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is a schematic diagram of the TGF-β₁ mRNA showing the boxedcoding sequence. The 112 amino acid TGF-β₁ (dashed) is encoded by the 3'end of the coding sequence. The sequenced cDNA inserts of λβCl, 3.19,3.32, 4.10, 4.33, 4.37 and 5.7b (described infra) and the genomic DNAsequence for the 3' untranslated region are aligned above the diagram.

FIGS. 1b(I)-1b(III) (hereinafter referred to collectively as FIG. 1b)depict the sequence and deduced amino acid sequence of the preTGF-β₁cDNA, determined from the several overlapping cDNAs and the genomic 3'region. The 5' terminal region which could be folded into stable hairpinloops is underlined. The preTGF-β₁ cDNA encodes a 390 amino acidprotein, of which the C-terminal 112 amino acids (boxed) encode matureTGF-β₁. A hydrophobic domain found at the N-terminus of the precursor isoverlined. An overlined Arg-Arg dipeptide precedes the proteolyticcleavage site for release of TGF-β₁. Three potential N-glycosylationsites in preTGF-β₁ are overlined. The stop codon is followed by theunderlined G-C rich sequence and a downstream TATA-like sequence.

FIG. 2 depicts a genomic fragment encoding a TGF-β₁ exon and its deducedamino acid sequence. Arrows show the mRNA processing sites (intron-exonjunctions). The residue numbers correspond to FIG. 1b.

FIG. 3 is a comparison between the known N-terminal sequence of bovineTGF-β₂ and the mature human TGF-β₁ and human and porcine TGF-β₃ aminoacid sequences. Unconserved substitutions in TGF-β₁ which constitute theTGF-β₂ and β₃ subtypes are designated by dots above the unconservedresidues. The amino acid residue numbers shown in this Figure shall beused herein unless otherwise indicated.

FIGS. 4a-4c show the cDNA sequences for human and porcine TGF-β₃. Thesequence for the human cDNA encodes a portion of the presequence regionand all of the mature sequence. The human and porcine sequences areadjacent to the lines designated hu4 and 10+11.3, respectively. Gaps areintroduced into the sequences in order to maximize nucleotide homology.Homologous bases are designated with an asterisk.

FIG. 5 depicts the amino acid sequences encoded by the cDNAs of FIGS.41-4c. Homologous residues are designated with an asterisk. Candidatetranslational start methionyl residues for the porcine sequence arelocated at positions 43, 88 or 90 (boxed methionyl residues). TheC-terminal residue for both the human and porcine sequences is the serylat 499 (porcine) or 204 (human).

DETAILED DESCRIPTION

TGF-β has proven to be extremely difficult to synthesize in recombinantcell culture while retaining its growth-altering activity. As can beseen from FIGS. 1b, 3 and 5, the mature TGF-β amino acid sequencecontains a large number of cysteine residues⁹, at least some of whichapparently are involved in interchain crosslinking in forming thehomodimeric TGF-β which is recovered from natural sources. Furthermore,TGF-β is expressed as a precursor molecule having a large amino terminalregion not containing the recognizable NH₂ -terminal signal peptidesequence typical of most secreted proteins, even through TGF-β normallyappears to some degree in the extracellular medium. However, eukaryoticcells have been transformed to express heterologous TGF-β,notwithstanding the anticipated difficulty in properly processing theprimary translation product in recombinant culture.

This invention is directed to recombinant synthesis of TGF-β, which isdefined as inclusive of biologically active preTGF-β having the FIG. 1bsequence, mature TGF-β, polypeptide fragments thereof and insertion,substitution and/or deletion variants of such preTGF-β (includingalleles or other TGF-β subtypes than the TGF-β₁ subtype shown in FIG.1b), mature TGF-β or polypeptide fragments.

Biologically active TGF-β is defined as being capable of inducingEGF-potentiated anchorage independent growth of target cell lines⁸¹and/or growth inhibition of neoplastic cell lines²³. Anchorageindependent growth refers to the ability of TGF-β and EGF treatednon-neoplastic target cells to form colonies in soft agar, acharacteristic ascribed to transformation of the cells (hence the nametransforming growth factor). This is not to say that TGF-β will "cause"cancer because it is now known that many normal cells express TGF-β.Paradoxically, TGF-β also is known to inhibit the growth of severalnormal cell types and various neoplastic cells such as A549.

Biological activity for the purposes herein also generally includes theability to cross-react with antisera raised against native TGF-β. NativeTGF-β is that which is obtained from platelets or other natural sources.Immunological cross-reactivity is a measure of a single active epitopeand not necessarily of active TGF-β domains involved in inducinganchorage-independent growth of target cells. However, immunologicallycross-reactive proteins per se are not biologically active as definedherein unless they also exert growth-affecting activity, i.e., allbiologically active TGF-β variants promote growth in the defined assays,but not all immunologically cross-reactive TGF-βs are biologicallyactive. Of course, TGF-β which is capable of inducing anchorageindependent growth frequently will exhibit immunologicalcross-reactivity with antisera raised against the native molecule as acorollary to maintenance of proper conformation.

The FIG. 1b nucleotide sequence was obtained by an analysis of severaloverlapping cDNAs and gene fragments, leading to the determination of acontinuous sequence corresponding to the TGF-β₁ precursor mRNA.According to FIG. 1a an initiator ATG is located 841 nucleotides fromthe 5' end and establishes a coding sequence for a 390 residuepolypeptide. Several areas within the cDNA sequence have anexceptionally high G-C content. The initiator ATG is flanked by two G-Crich areas of approximately 200 bp each. In addition, several regions ofthe cDNA, particularly the 5'-terminus, have regions with greater than80 percent G-C content. The location of these G-C rich regions coincideswith the areas in which the many cDNA cloning artifacts occurred andwhere partial length cDNAs were obtained.

The 5' untranslated region of the TGF-β₁ is 841 nucleotides long(assuming the ATG is located at nucleotide 842) and contains a longsequence consisting almost exclusively of purines. The biologicalrelevance of this exceptionally long 5' untranslated region of high G-Ccontent is unknown, but it is similar to the structural organization ofc-myc mRNA. However, there is no striking sequence homology betweenthese two sequences. The long 5' untranslated region of c-myc has beenhypothesized to have a functional significance³⁷. The G-C rich5'-proximal part of the 5' untranslated sequence of human c-myc mRNA hasseveral regions which could form stable hairpin loops. Likewise, thefirst 120 bp of the untranslated preTGF-β₁ cDNA can theoretically befolded into hairpin loop structures with a calculated stability of -91kcal. The long 5' untranslated sequence and the potentially stablehairpin loop structures could play a role in the mRNA stability or inthe regulation of transcription. Accordingly, this region can be deletedand substituted for by other 5' untranslated sequences, e.g. from viralproteins, in order to identify structures that may improve TGF-β₁ yieldsfrom recombinant cell culture.

The stop codon preceding base 2015 is immediately followed by aremarkable, G-C rich sequence of 75 nucleotides (underlined in FIG. 1b).This sequence consists of multiple repeats of CCGCC. The peculiar natureof this sequence is probably responsible for the fact that the 3'untranslated end of the mRNA could not be cloned as a cDNA sequence,perhaps due to the inability of the E. coli DNA polymerase I to use thissequence of a similar nature have been found in the promoter regions ofthe genes for human dihydrofolate reductase³⁸, human transferrinreceptor, human adenosine deaminase³⁹, and Herpesvirus thymidinekinase⁴⁰. In the latter case, McKnight et al.⁴⁰ have shown that thesestructural elements are of major importance for the transcriptionefficiency. In addition, it has been shown that the promoter specifictranscriptional factor Spl binds to such sequences in the SV40 earlypromoter region and in a related monkey promoter⁴¹,42. In all of thesecases the G-C rich repeats are followed closely by the Goldberg-HognessTATA sequence. In the case of preTGF-β₁, however, these sequences arelocated in the 3' untranslated region of the gene, but are interestinglyalso followed by a TATA-like sequence. No evidence that this regioncould function as a promoter is available. The preTGF-β₁ gene sequencehas the hexanucleotide AATAAA about 500 nucleotides downstream from thestop codon. This sequence, which usually precedes the site ofpolyadenylation by 11 to 30 bases³², probably functions as the preTGF-β₁mRNA polyadenylation signal, since this would be in agreement with thesize of preTGF-β mRNA estimated from Northern hybridizations, and since3' untranslated regions rarely contain intervening sequences. Benoist etal.⁴³ have proposed a consensus sequence TTCACTGC which follows theAATAAA closely and immediately precedes the polyA-tail. A similarsequence, TTCAGGCC, follows the AATAAA sequence in the 3' untranslatedregion of the preTGF-β₁ mRNA, providing further support for theassignment of the polyadenylation site at position 2530 (FIG. 1b).

PreTGF-β₁ is a polypeptide of 390 amino acids. Comparison of thissequence with the previously determined NH₂ -terminus of mature TGF-β₁shows that TGF-β₁ constitutes the C-terminal 112 amino acids ofpreTGF-β₁. The mature TGF-β₁ monomer is cleaved from the precursor atthe Arg-Arg dipeptide immediately preceding the mature TGF-β₁ NH₂-terminus. A similar dibasic cleavage site is located immediatelyupstream from the mature TGF-β₃ amino terminus. Such proteolyticcleavage sites have been found in several other polypeptide precursorsequences, including preproenkephalin⁴⁴,45, the calcitonin precursor⁴⁶,and corticotropin-β-lipotropin precursor⁴⁷. Determination of thehydrophobicity profile by the method of Kyte and Doolittle⁴⁸ predictsthat the Arg-Arg sequence is located within a hydrophilic region whichwould make it accessible to a trypsin-like peptidase. Post-translationalcleavage of the precursor gives rise to the mature TGF-β monomer. Thedisposition of the presequence is not known but may give rise to otherbiologically active peptides. The TGF-β₁ and TGF-β₃ precursors containseveral pairs of basic residues (FIGS. 1b and 5) which could alsoundergo post-translation cleavage and give rise to separate polypeptideentities. Mature TGF-β₁ contains two Arg-Lys dipeptides which apparentlyare not cleaved. As shown in FIG. 1b, the preTGF-β₁ precursor containsthree potential N-glycosylation sites, Asn-X-Ser or Thr (FIG. 1b). Noneof these are localized within mature TGF-β₁. Accordingly, a method isprovided whereby mature TGF-β is purified free of glycoproteins byadsorbing the glycoproteins on immobilized lectins and eluting TGF-βwith the unadsorbed fraction.

The sequence for human TGF-β was determined by direct amino acidsequence analysis and by deduction from the TGF-β cDNA. The sequence ofthe different TGF-β₁ peptides obtained by clostripain digestion is inagreement with the cDNA sequence, except for a few residues whichpresumably are due to incorrect amino acid assignment in sequencing. The112 amino acid TGF-β sequence contains 9 cysteines, whereas the rest ofthe precursor contains only two (FIGS. 1b and 5). Previous studies haveshown that reduction of the TGF-β₁ dimer of 25 kd results in thegeneration of two polypeptide chains of 12.5 kd¹⁵. Sequence analysis ofthe TGF-β amino-terminus and of the TGF-β₁ peptides obtained afterclostripain digestion strongly suggests that the TGF-β dimer consists oftwo identical polypeptides. This homodimeric nature is also supported bythe presence of only a single hybridizing DNA fragment upon Southernhybridization of human genomic DNA with a TGF-β exon probe. Chou-Fasmananalysis⁵⁰ of the secondary structure shows that the TGF-β polypeptidehas an extensive β -sheet character with little, if any, α-helicity. Theregion immediately preceding the basic dipeptide cleavage site is likelyin an α-helical configuration.

For purposes herein, preTGF-β is defined as the normal TGF-β precursordepicted in FIGS. 1b and 5 as well as other precursor forms of TGF-β inwhich the presequence is not that normally associated with TGF-β. Theselatter forms are to be considered insertional mutants of DNA encodingmature TGF-β. These mutants ordinarily comprise a presequence which isheterologous to TGF-β in the form of a fusion with mature TGF-β. Theheterologous presequences preferably are obtained from other secretedproteins, for example pregrowth hormone, preproinsulin, viral envelopeproteins, interferons and yeast or bacterial presequences recognized bymammalian host cells. The sequences for these secretory leaders areknown, as are suitable sources for DNA encoding same if it is notdesired to synthesize the DNA in vitro. They are linked to DNA encodingmature TGF-β by restriction enzyme digestion of the DNA containing thedesired signal and the preTGF-β DNA. Synthetic oligonucleotides areprepared in order to introduce unique restriction sites (linkers) and,if necessary, DNA fragments needed to complete any presequence andmature TGF-β coding regions removed during restriction enzyme digestion.The synthesized linkers and/or fragments then are ligated to therestriction enzyme digest fragments containing the substitute signal andTGF-β coding region, and inserted into a cloning vector and the vectoris used to transform bacterial hosts. The mutant presequence thereafteris cloned into an expression vector and used to transform host cells. Anillustrative example employing a viral envelope protein presequence isdescribed below.

Optimally, DNA encoding the complete heterologous presequence is linkedto the first codon of TGF-β DNA. Alternatively, DNA encoding the matureTGF-β coding sequence is ligated to DNA encoding the completeheterologous presequence plus a short portion, e.g. 21 to 45 base pairs,encoding the mature heterologous protein; this will result in thesecretion of a fusion peptide or protein which is useful as an immunogenor which can be cleaved to yield mature TGF-β (for example, by insertionof a collagenase cleavage site between the N-terminus of TGF-β and theC-terminus of the heterologous protein fragment). The objective of theseconstructions is to substitute a high efficiency secretory system forthe native TGF-β secretory leader. However, it is by no means necessaryto secrete TGF-β in order to produce it in recombinant culture.

Other deletion-insertion mutants include linking mature TGF-β species toviral proteins expressed in large intracellular quantities, e.g.retroviral core proteins, large T antigen from SV40 and the like, or toimmunogenic bacterial proteins or polypeptides such as chemotacticpolypeptides, in particular the potent chemotactic tripeptideMet-Leu-Phe-.

Expressed variants of preTGF-β, mature TGF-β or fragments thereof willexhibit amino acid sequences that gradually depart from the FIGS. 1b or5 sequences as the number and scope of insertions, deletions andsubstitutions increases. This departure is measured as a reduction inhomology between preTGF-β and the variant. All proteins or polypeptidesthat display TGF-β anchorage independent growth-promoting biologicalactivity are included within the scope of this invention, regardless ofthe degree of homology that they show to the FIG. 1 protein. The reasonfor this is that some regions of preTGF-β, e.g. the presequence, arereadily mutated, or even completely deleted as in the case of matureTGF-β, and thus biological activity will be retained. On the other hand,deletion of the nine cysteine residues (and accompanying disulfidelinkages) in the mature TGF-β molecule will have a substantial adverseimpact on this biological activity and in all likelihood wouldcompletely abrogate biological activity. In addition, a substitutionmutant may exhibit full TGF-β growth-promoting activity and yet be lesshomologous if residues containing functionally similar amino acid sidechains are substituted. Functionally similar refers to dominantcharacteristics of the side chains such as hydrophobic, basic, neutralor acidic, or the presence or absence of steric bulk. Thus the degree ofhomology that a given polypeptide bears to preTGF-β is not the principalmeasure of its identity as TGF-β. However, as a general guide, proteinsor polypeptides that share at least some biological activity with matureTGF-β from natural sources and which are substantially homologous withthe FIG. 1b sequence are to be considered as falling within the scope ofthe term TGF-β, e.g., TGF-β variants being about from 40 percent to 100percent homologous with preTGF-β or any fragment thereof greater thanabout 20 residues, including variants having an amino acid sequencegreater than about 75 percent homologous with the mature TGF-β₁sequence. With respect to neoplastic cell growth-inhibiting activity,TGF-β excludes polypeptides known heretofore to exert such growthinhibitory activity, e.g. interferons, tumor necrosis factor andlymphotoxin, but otherwise need not necessarily have homologous regionswith the FIG. 1b sequences.

More narrow and specific factors in establishing the identity of apolypeptide as TGF-β are (a) the ability of antisera which are capableof substantially neutralizing the growth inhibitory or the anchorageindependent growth promoting activity of mature TGF-β also tosubstantially neutralize the activity of the polypeptide in question, or(b) the ability of the candidate polypeptide to compete with TGF-β for aTGF-β cell surface receptor. However, it will be recognized thatimmunological identity and growth promoting identity are not necessarilycoextensive. A neutralizing antibody for the mature TGF-β of FIG. 1b maynot bind a candidate protein because the neutralizing antibody happensto not be directed to a site on TGF-β that is critical for its growthpromoting activity. Instead, the antibody may bind an innocuous regionand exert its neutralizing effect by steric hindrance. Therefore, acandidate protein mutated in this innocuous region might no longer bindthe neutralizing antibody, but it would nonetheless be TGF-β in terms ofsubstantial homology and biological activity.

The TGF-β residues which are subject to site-directed mutagenesis forthe preparation of variants which are likely to be antagonists tobiologically active TGF-β are the cysteine residues, Arg₁₈, Lys₁₉,Leu₂₀, Tyr₂₁, Ile₂₂, Phe₂₄, Leu₂₈, Gly₂₉, Trp₃₀, Trp₃₂, Ile₃₃, Pro₃₆,Gly₃₈, Tyr₃₉, Asn₄₂, Gly₄₆, Pro₄₉, Leu₆₂, Tyr₆₅, Pro₇₀, Val₇₉, Pro₈₀,Leu₈₃, Leu₈₆, Ile₈₉, Val₉₀, Tyr₉₁, Tyr₉₂, Leu₁₀₂, Asn₁₀₅, Met₁₀₆, Ile₁₀₇and Val₁₀₈. Substitutions which are made at these residues generallywill be non-conserved, i.e. the substituted residue (a) differssubstantially in hydrophobicity, for example a hydrophobic residue (Val,Ile, Leu, Phe or Met) substituted for a hydrophilic residue such as Arg,Lys, Trp or Asn, or a hydrophilic residue such as Thr, Ser, His, Gln,Asn, Lys, Asp, Gly or Trp substituted for a hydrophobic residue; (b)differs substantially in its effect on polypeptide backbone orientationsuch as substitution of or for Pro or Gly by another residue; (c)differs substantially in electric charge, for example substitution of anegatively charged residue such as Gly or Asp for a positively chargedresidue such as Lys, His or Arg (and vice versa); or (d) differssubstantially in steric bulk, for example substitution of a bulkyresidue such as His, Trp, Phe or Tyr for one having a minor side chain,e.g. Ala, Gly or Ser (and vice versa). Each of the foregoing targetresidues also is deleted (preferably in pairs) or non-conserved residues(also preferably in pairs) are inserted adjacent to the target residues.Ordinarily, only one residue at a time is subject to introduction ofsequence variation. The regions for investigation of site-directedvariation are residues 105-112, 77-95, and 20-49.

Identification of antagonists is routine. The candidate is incubatedtogether with an equimolar amount of TGF-β otherwise detectable in theEGF-potentiated anchorage independent target cell growth assay⁸¹, andthe culture observed for failure to induce anchorage independent growth.

Antagonists that remain immunologically cross-reactive with native TGF-βare useful in immunoassays as standards or, when labelled, as reagentsin competitive-type assays. Antagonists also are useful in therapy, e.g.of TGF-β dependent tumors.

The same residues targeted for site-directed variation in the generationof candidate antagonists also are targeted for the generation ofagonists. Here, however, the variant residues which are substituted orinserted are conserved, i.e., members of each class, e.g. hydrophobic,electronegative and the like as described above, are substituted for oneanother or inserted adjacent to a member of the same class, againpreferably in pairs.

Also within the scope of this invention are TGF-β variants representingfusions of one TGF-β allele or sub-type with another, for example aC-terminal domain from TGF-β₁ having the homologous N-terminal domainsubstituted from TGF-β₃, or substitutional variants in which a domainfrom one allele or subtype is substituted for the homologous region fromanother allele or subtype.

It is important to observe that characteristics such as molecular weightand the like for the native or wild type mature TGF-β of FIG. 1aobtained from placenta or platelets are descriptive only for the nativespecies of TGF-β. The variants contemplated herein may modify thecharacteristics of native TGF-β considerably, and this in fact may bethe objective of the mutagenesis as is more fully described below. WhileTGF-β as defined herein includes native TGF-β₁, other relatedbiologically active polypeptides will fall within the definition. TGF-βspecies like the insertion mutants, deletion mutants, or fusion proteinsdescribed above will bring the mutant outside of the molecular weightestablished for native TGF-β. For example, fusion proteins with matureTGF-β or preTGF-β itself will have a greater molecular weight thannative, mature TGF-β, while deletion mutants of mature TGF-β will have alower molecular weight. Similarly, TGF-β is engineered in order tointroduce glycosylation sites, thereby resulting in glycosylated TGF-β,or to substitute serine for cysteine at sites not critical forbiological activity. Finally, post-translational processing of humanpreTGF-β in cell lines derived from nonprimate mammals may producemicroheterogeneity in the amino terminal region of mature TGF-β, so thatalanine will no longer by the amino terminal amino acid.

Note that the language "capable" of inducing anchorage independentgrowth in the definition for biological activity means that the preTGF-βor fragments thereof include polypeptides which can be converted, as byenzymatic digestion, to a polypeptide fragment which exhibits thedesired biological activity. Typically, inactive precursors will befusion proteins in which mature TGF-β is linked by a peptide bond at itscarboxyl terminus to an insoluble or gelatinous protein. The sequence ator within the region of this peptide bond is selected so as to besusceptible to proteolytic hydrolysis whereby TGF-β is released, eitherin vivo for in situ generation or, as part of a manufacturing protocol,in vitro.

While TGF-β ordinarily means human TGF-β, TGF-β from sources such asmurine, porcine, equine or bovine is included within the definition ofTGF-β so long as it otherwise meets the standards described above forbiological activity. TGF-β is not species specific, e.g., murine andhuman TGF-β are both effective in inducing anchorage independent growthof the same cell line. Therefore, TGF-β from one species can be used intherapy of another species. DNA encoding the TGF-β of other species isobtained by probing cDNA or genomic libraries from such species withlabelled human preTGF-β cDNA.

Derivatives of TGF-β are included within the scope of this invention.Derivatives include glycosylated and covalent or aggregative conjugateswith other TGF-β molecules, dimers or unrelated chemical moieties.Covalent derivatives are prepared by linkage of functionalities togroups which are found in the TGF-β amino acid chains or at the N- orC-termini, by means known in the art. These derivatives may, forexample, include: aliphatic or acyl esters or amides of the carboxylterminus, alkylamines or residues containing carboxyl side chains, e.g.,conjugates to alkylamines at aspartic acid residues; O-acyl derivativesof the amino terminal amino acid or amino-group containing residues,e.g. conjugates with fMet-Leu-Phe or immunogenic proteins. Derivativesof the acyl groups are selected from the group of alkyl-moieties(including C3 to C10 normal alkyl), thereby forming alkanoyl species,and carbocyclic or heterocyclic compounds, thereby forming aroylspecies. The reactive groups preferably are difunctional compounds knownper se for use in cross-linking proteins to insoluble matrices throughreactive side groups.

Covalent or aggregative derivatives are useful as reagents inimmunoassay or for affinity purification procedures. For example, TGF-βis insolubilized by covalent bonding to cyanogen bromide-activatedSepharose by methods known per se or adsorbed to polyolefin surfaces(with or without glutaraldehyde cross-linking) for use in the assay orpurification of anti-TGF-β antibodies or cell surface receptors. TGF-βalso is labelled with a detectable group, e.g., radioiodinated by thechloramine T procedure, covalently bound to rare earth chelates orconjugated to another fluorescent moiety for use in diagnostic assays,especially for diagnosis of TGF-β levels in biological samples bycompetitive-type immunoassays.

TGF-β variants generally are made by predetermined, i.e. site specific,methods, The objective of site specific mutagenesis is to construct DNAthat encodes TGF-β as defined above, i.e., TGF-β which exhibitsbiological activity.

While the mutation site is predetermined, it is unnecessary that themutation per se be predetermined. For example, in order to optimize theperformance of the mutants at a given position random mutagenesis isconducted at the target codon and the expressed TGF-β variants arescreened for optimal activity. Techniques are well known for makingsubstitution variants at predetermined sites in DNA having a knownsequence, for example, M13 primer mutagenesis.

TGF-β mutagenesis is conducted by making amino acid insertions, usuallyon the order of about from 1 to 5 amino acid residues, or deletions ofabout from 1 to 10 residues. Substitutions, deletions, insertions or anysubcombination may be combined to arrive at a final construct. As notedabove, insertions include amino or carboxyl-terminal fusions, e.g. witha hydrophobic or immunogenic protein. The mutations in the DNA encodingsuch mutations should not ultimately plate the sequence out of readingframe in an expression vector whereby the resulting protein is notbiologically active TGF-β. The mutations also preferably will not createcomplementary regions that could produce translation-suppressingsecondary mRNA structure.

Included herein are heterodimers of TGF-β. These typically includedimers in which one TGF-β chain is from one subclass, e.g. TGF-β₁, whilethe other is from another subclass, e.g. TGF-β₃. Similarly, TGF-β aminoacid sequence variants are produced as homodimers or heterodimers withother amino acid sequence variants or with native TGF-β sequences.Heterodimers include dimers containing a first TGF-β sequence that isbiologically active when present in a homodimer and a second TGF-βsequence that is not biologically active. Such heterodimers may beactive as TGF-β antagonists. Heterodimers are readily produced bycotransforming host cells with DNA encoding the TGF-β chains selected. Aproportion of the TGF-β so produced is expressed as the desiredheterodimer, and transformants are screened for those which produce thegreatest proportion of heterodimer.

DNA which encodes TGF-β is obtained by chemical synthesis, by screeningreverse transcripts of mRNA from placental or other cells or byscreening genomic libraries from eukaryotic cells. This DNA need not usethe codons set forth in FIGS. 1b or 4a-4c so long as the host cellrecognizes the codons which are used. DNA of this sort is as easilymanufactured in vitro as the DNA of FIGS. 1b or 4a-4c. Also usefulherein is nucleic acid, either RNA or DNA, which does not encode TGF-βthereof as defined herein but which nonetheless is capable ofhybridizing with such DNA or RNA. Noncoding but hybridizing nucleicacid, while not used in the recombinant synthesis of TGF-β, is useful asan intermediate for making labelled probes in diagnostic assays forTGF-β mRNA or genomic DNA in test cells.

Diagnostic nucleic acid is covalently labelled with a detectablesubstance such as a fluorescent group, a radioactive atom or achemiluminescent group by methods known per se. It is then used inconventional Southern or Northern hybridization assays. Such assays areemployed in identifying TGF-β vectors and transformants as described inthe Examples infra, or for in vitro diagnosis such as detection of TGF-βmRNA in tissues as a measure of mitogenic activity.

TGF-β is synthesized herein in host cells transformed with vectorscontaining DNA encoding TGF-β. A vector is a replicable nucleic acidconstruct. Vectors are used either to amplify and/or to express DNAwhich encodes TGF-β, or to amplify DNA that hybridizes with DNA or RNAencoding TGF-β. An expression vector is a replicable DNA construct inwhich a DNA sequence encoding TGF-β is operably linked to suitablecontrol sequences capable of effecting the expression of TGF-β in asuitable host. Such control sequences include a transcriptionalpromoter, an optional operator sequence to control transcription, asequence encoding suitable mRNA ribosomal binding sites, and sequenceswhich control termination of transcription and translation. Forexpression of TGF-β in eukaryotic cells the vector also should includeDNA encoding a selection gene. However, the selection gene can besupplied by an unlinked plasmid in cotransformation.

Vectors comprise plasmids, viruses (including phage), and integratableDNA fragments i.e., fragments that are integratable into the host genomeby recombination. In the present specification, the vector is a plasmidin the sense that it is cloned in bacterial cells, but it integratesinto the host cell genome upon cotransformation. However, all otherforms of vectors which serve an equivalent function and which are, orbecome, known in the art are suitable for use herein. Suitable vectorswill contain replicon and control sequences which are derived fromspecies compatible with the intended expression host.

DNA regions are operably linked when they are functionally related toeach other. For example, DNA for a presequence or secretory leader isoperably linked to DNA for a polypeptide if it is expressed as apreprotein which participates in the secretion of the polypeptide; apromoter is operably linked to a coding sequence if it controls thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to permittranslation. Generally, operably linked means contiguous and, in thecase of secretory leaders, contiguous and in reading phase.

Cultures of cells derived from multicellular organisms are the preferredhost cells herein. In principle, any higher eukaryotic cell culture isworkable, whether from vertebrate or invertebrate culture. However,interest has been greatest in invertebrate cells, However, interest hasbeen greatest in invertebrate cells, and propagation of vertebrate cellsin culture per se has become a routine procedure in recent years [TissueCulture, Academic Press, Kruse and Patterson, editors (1973)]. Examplesof useful host cell lines are VERO and HeLa cells, Chinese hamster ovary(CHO) cell lines, and WI38, BHK, COS-7 293 and MDCK cell lines.

Many eukaryotic cells are known to synthesize endogenous TGF-β. Thus,many potential host cells synthesize TGF-β of the host species. ThisTGF-β therefore is present in the TGF-β produced by transcription andtranslation of the transforming DNA. For example, hamster TGF-β ispresent in transformed CHO cells, and thus is present in cell extractscontaining human TGF-β harvested from such cells. While this is notnecessarily disadvantageous because animal and human TGF-β is activeacross species lines, it is desirable to select a host/vector systemthat secretes as little of the endogenous animal TGF-β as possible. Thisis accomplished by (a) selecting host animal cell lines that synthesizelow levels of animal TGF-β, (b) transforming the animal cell line with avector for high efficiency TGF-β secretion (described above) andrecovering human TGF-β from the culture medium or (c) transforming ahuman cell line, whereby any endogenous hTGF-β that is produced wouldnot be a contaminant.

It may be desirable to use a host cell line that is differentiated tosynthesize endogenous TGF-β. Examples include megakaryoblast,promegakaryocytic or basophilic megakaryocytic cell lines. If suitablecell lines are not available they may be produced by EBV immortalizationof megakaryoblasts, promegakaryocytes or basophilic megakaryocytesrecovered from mammalian bone marrow. The TGF-β of the desired speciesis recovered from transformant cell cultures by immunoaffinitychromatography using antibodies specific for host TGF-β.

Expression vectors for such cells ordinarily include an origin ofreplication (for extrachromosomal amplification), a promoter locatedupstream from the TGF-β coding sequences, along with an enhances ifdesired, RNA splice site (if intron-containing TGF-β-encoding genomicDNA is used), and a transcriptional termination sequence including apolyadenylation site located 3' to the TGF-β sequence.

The transcriptional and translational control sequence in expressionvectors to be used in transforming vertebrate cells preferably areprovided from viral sources. For example, commonly used promoters arederived from polyoma, Adenovirus 2, and most preferably Simian Virus 40(SV40). The early and late promoters of SV40 are particularly usefulbecause both are obtained easily from the virus as a fragment which alsocontains the SV40 viral origin of replication ⁵⁴. Smaller or larger SV40fragments may also be used, provided the approximately 250 bp sequenceextending from the Hind III site toward the Bgl I site located in theviral origin of replication is included. Since TGF-β appears to be toxicto mammalian cell transformants and thus may interfere with attempts toamplify the gene, yields may be improved by the use of induciblepromoters, e.g. the metallothionein promoter, Drosophila heat shockpromoter or mouse mammary tumor virus promoter. Further, it is alsopossible to utilize the TGF-β genomic promoter, control and/or signalsequences normally associated with TGF-β, provided such controlsequences are compatible with and recognized by the host cell. If TGF-βuntranslated regions are included in expression vectors, yields may beimproved by substituting A or T bases for G or C bases immediately 5' tothe start codon and deleting G-C rich domains in the 3' untranslatedsequences of the cDNA.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral (e.g. Polyoma, Adenovirus, VSV, or BPV) source, or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient.

Rather than using vectors which contain viral origins of replication,one can transform mammalian cells by cotransformation with a selectablemarker and the TGF-β DNA. An example of a suitable selectable marker isdihydrofolate reductase (DHFR) or thymidine kinase. Such markers areproteins, generally enzymes that enable the identification oftransformant cells, i.e., cells which had been competent to take upexogenous DNA. Generally, identification is by survival of transformantsin culture medium that is toxic or from which the cells cannot obtaincritical nutrition without having taken up the marker protein. Inselecting a preferred host mammalian cell for transfection by vectorswhich comprise DNA sequences encoding both TGF-β and DHFR, it isappropriate to select the host according to the type of DHFR proteinemployed. If wild type DHFR protein is employed, it is preferable toselect a host cell which is deficient in DHFR thus permitting the use ofthe DHFR, coding sequence as a marker for successful transfection inselective medium which lacks hypoxanthine, glycine, and thymidine. Anappropriate host cell in this case is the Chinese hamster ovary (CHO)cell line deficient in DHFR activity, prepared and propagated asdescribed by Urlaub and Chasin⁵⁵.

On the other hand, if DNA encoding DHFR protein with low bindingaffinity for methotrexate (MTX) is used as the controlling sequence, itis not necessary to use DHFR resistant cells. Because the mutant DHFR isresistant to MTX, MTX containing media can be used as a means ofselection provided that the host cells are themselves MTX sensitive.Most eukaryotic cells which are capable of absorbing MTX appear to bemethotrexate sensitive. One such useful cell line is a CHO line, CHO-Kl(ATCC No. CCL 61). Alternatively, DHFR⁺ host cells are used bycotransforming the cells with DNA encoding the neomycin resistance gene,DHFR and TGF-β. The initial transfections are screened for neomycinresistance, and resistant transformants then amplified on MTX.

Other methods suitable for adaptation to the synthesis of TGF-β inrecombinant vertebrate cell culture are described in Gething et al.⁵⁶,Mantei et al.⁵⁷, and Levinson et al.⁵⁸,59.

TGF-β is recovered from lysed, transformed cells and insoluble celldebris separated by centrifugation. Alternatively, the culturesupernatants from transformed cells that secrete TGF-β are simplyseparated from the cells by centrifugation. Then the TGF-β generally ispurified by methods known in the art¹⁵,16,17 using gel filtration in thepresence of acid followed by HPLC and elution on an acetonitrilegradient. However, such methods are not necessarily required to preparea therapeutic product.

As a further or substitute purification step, cell lysates orsupernatants are heated for a period and at a temperature sufficient todenature and precipitate contaminant proteins but not TGF-β; TGF-β is aremarkably heat stable protein, perhaps as a result of extensivedisulfide bond formation. As a result, the heating should be conductedin a medium that contains low amounts of disulfide reagents such asdithiothreitol or the like. Heating also is combined with acidificationsince TGF-β is known to be stable to 1M acetic acid.

Mature, native TGF-β is not glycosylated. Therefore it is separated fromany residual contaminant heat- and acid-stable glycoproteins byadsorbing the glycoproteins on lectin columns such as lentillectin-linked sepharose. This step, less desirably, can go before theheat and acid treatment. TGF-β will elute with the unadsorbed fraction.The recombinant TGF-β is recovered from host cells expressing endogenousTGF-β (or undesired homodimers) by transforming the host cells with aTGF-β variant which is glycosylated by the host. The sugar "tag" enablesthe recombinant TGF-β to be recovered free of endogenous TGF-β by lectinaffinity chromatography, elution of the glycosylated TGF-β and removal,if desired, of the sugar residues by conventional enzymatic digestion.

If high purity product is desired the crude or partially purifiedmixture thereafter is subjected to chromatofocusing.

TGF-β is prepared for administration by mixing TGF-β at the desireddegree of purity with physiologically acceptable carriers, i.e.,carriers which are nontoxic to recipients at the dosages andconcentrations employed. Ordinarily, this will entail combining TGF-βwith buffers, antioxidants such as ascorbic acid, low molecular weight(less than about 10 residues) polypeptides, proteins, amino acids,carbohydrates including glucose or dextrins, chelating agents such asEDTA, and other excipients. TGF-β for use in therapeutic administrationmust be sterile. This is readily accomplished by filtration throughsterile filtration (0.2 micron) membranes. TGF-β ordinarily will bestored as an aqueous solution since it is highly stable to thermal andoxidative denaturation.

TGF-β optionally is combined with activating agents such as TGF-α or EGFspecies as is described further in U.S. Ser. No. 500,833, now abandonedand is administered in accord with said application.

Various therapeutic indications for TGF-β compositions are known.

The first, and preferred, indication is topical application to incisionsor exposed tissue for the promotion of wound healing. There are nolimitations as to the type of wound or other traumata that can betreated, and these include (but are not limited to): first, second andthird degree burns (especially second and third degree); epidermal andinternal surgical incisions, including those of cosmetic surgery;wounds, including lacerations, incision, and penetrations; and epidermalulcers including decubital (bed-sores), diabetic, dental, hemophiliac,and varicose. Doses such as those previously described for woundhealing¹⁷ will be suitable as starting doses in these indications.

TGF-β compositions are applied to burns in the form of a sterileirrigant, preferably in combination with a physiological salinesolution, or in the form of ointments or suspensions, preferably incombination with purified collagen. The compositions also may beimpregnated into transdermal patches, plasters, and bandages, preferablyin a liquid or semi-liquid form. Automicrobial agents such as silversulfadiazine should be included in such articles or compositions.Debridement agents such as proteolytic enzymes also can be included ifthey do not hydrolyze TGF-β or a hydrolysis-resistant TGF-β mutant isemployed.

TGF-β also is administered systemically for the treatment of wounds andsimilar traumata. Systemic administration is useful provided that thereare no, or limited, undesirable side-effects, such as the stimulation ofneoplastic cellular growth in patients with cancer. TGF-β compositionsfor systemic administration preferably are formulated as sterile,isotonic parenteral injections or infusions.

The amount of activating agent (such as TGF-α, EGF or other growthfactors) administered with TGF-β depends directly upon the amount ofTGF-β present in the activated compositions as administered to therecipient, the growth factors selected and the clinical status of thepatient.

Initial dosing of TGF-β should be delivered to the therapeutic site in aconcentration of about from 0.1 to 150 ng/ml and thereafter adjusted inline with clinical experience. Since TGF-β compositions both provoke andsustain cellular regeneration, a continual application or periodicreapplication of the compositions is indicated. The clinician will beexpected to modify the dosage in accordance with clinical experience.

In order to simplify the Examples certain frequently occurring methodswill be referenced by shorthand phrases.

Plasmids are designated by a low case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein arecommercially available, are publicly available on an unrestricted basis,or can be constructed from such available plasmids in accord withpublished procedures. In addition, other equivalent plasmids are knownin the art and will be apparent to the ordinary artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with anenzyme that acts only at certain locations in the DNA. Such enzymes arecalled restriction enzymes, and the sites for which each is specific iscalled a restriction sites. The various restriction enzymes used hereinare commercially available and their reaction conditions, cofactors andother requirements as established by the enzyme suppliers were used.Restriction enzymes commonly are designated by abbreviations composed ofa capital letter followed by other letters representing themicroorganism from which each restriction enzyme originally was obtainedand then a number designating the particular enzyme. In generally, about1 μg of plasmid or DNA fragment is used with about 2 units of enzyme inabout 20 μl of buffer solution. Appropriate buffers and substrateamounts for particular restriction enzymes are specified by themanufacturer. Incubation times of about 1 hour at 37° C. are ordinarilyused, but may vary in accordance with the supplier's instructions. Afterincubation, protein is removed by extraction with phenol and chloroform,and the digested nucleic acid is recovered from the aqueous fraction byprecipitation with ethanol. Digestion with a restriction enzymeinfrequently is followed with bacterial alkaline phosphatase hydrolysisof the terminal 5' phosphates to prevent the two restriction cleavedends of a DNA fragment from "circularizing" or forming a closed loopthat would impede insertion of another DNA fragment at the restrictionsite. Unless otherwise stated, digestion of plasmids is not followed by5' terminal dephosphorylation. Procedures and reagents fordephosphorylation are conventional⁸⁵.

"Recovery" or "isolation" of a given fragment of DNA from a restrictiondigest means separation of the digest on polyacrylamide or agarose gelby electrophoresis, identification of the fragment of interest bycomparison of its mobility versus that of marker DNA fragments of knownmolecular weight, removal of the gel section containing the desiredfragment, and separation of the gel from DNA. This procedure is knowngenerally⁸⁶,87.

"Southern Analysis" is a method by which the presence of DNA sequencesin a digest or DNA-containing composition is confirmed by hybridizationto a known, labelled oligonucleotide or DNA fragment. For the purposesherein, unless otherwise provided, Southern analysis shall meanseparation of digests on 1 percent agarose, denaturation and transfer tonitrocellulose by the method of E. Southern⁶⁸, and hybridization asdescribed by T. Maniatis et al.⁸⁸.

"Transformation" means introducing DNA into an organism so that the DNAis replicable, either as an extrachromosomal element or chromosomalintegrant. Unless otherwise provided, the method used herein fortransformation of E. coli is the CaCl₂ method of Mandel et al.⁸⁹

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (T. Maniatis et al., Id., p.146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase ("ligase")per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

"Preparation" of DNA from transformants means isolating plasmid DNA frommicrobial culture. Unless otherwise provided, the alkaline/SDS method ofManiatis et al., Id., P. 90, may be used.

"Oligonucleotides" are short length single or double strandedpolydeoxynucleotides which are chemically synthesized by known methodsand then purified on polyacrylamide gels.

All citations are expressly incorporated by reference.

EXAMPLE 1 Purification and Sequence Analysis of Human TGF-β

The known purification method of Assoian et al.¹⁵ was scaled up andmodified to obtain enough homogeneously pure human TGF-β₁ for amino acidsequencing. 250 units of human platelets were extracted in a Waringblender with 1 liter of acid-ethanol. Addition of 4 liters of ether gaverise to a precipitate which was collected by vacuum filtration overWhatman No. 1 paper. The precipitate was dissolved overnight in 50 ml of1M acetic acid and purified by gel filtration on a Biogel P-60 column(10×100 cm), equilibrated in 1M acetic acid. The fractions containingTGF-β₁ were identified by analytical SDS-polyacrylamide gelelectrophoresis and bioassay¹⁵. Peak fractions were pooled, freeze-driedand redissolved in 20 ml 1M acetic acid, 8M urea. Subsequent gelfiltration over a Biogel P-60 column (5×90 cm) in 1M acetic acid, 8Murea yielded about 50 percent pure TGF-β. These peak fractions were thendiluted with 1 volume of water and applied to a semipreparative RPP C18(Synchropak) HPLC column in 0.1 percent trifluoroacetic acid and elutedwith a 20-50 percent acetonitrile gradient. The TGF-β₁ thus obtained wasquantitated by amino acid analysis, showing a yield of about 0.5 mg perpreparation. Denaturing SDS-polyacrylamide gel electrophoresis wasperformed as described⁶⁰. In agreement with previous work thenon-reduced TGF-β₁ migrated as a 25 kD protein in a SDS-polyacrylamidegel, while reduction with β-mercaptoethanol converted it into a 12.5 kDspecies. This suggested that TGF-β consists of two 12.5 kD polypeptidechains linked by intermolecular disulfide bridges¹⁵.

In order to obtain protein sequence information, the purified TGF-β₁ wasreduced, alkylated and subjected to amino-terminal sequence analysis.1.2 nmole of TGF-β was dialyzed into 8M urea and reduced by incubationin 0.1M Tris-HCl (pH 8.5), 10 mM dithiothreitol, 8M urea. Subsequentalkylation took place in the presence of 50 mM iodoacetate at roomtemperature in the dark. This reaction was terminated after 30 min. byaddition of an excess β-mercaptoethanol and dialysis. 0.7 nmole of thisTGF-β₁ was used for the direct NH₂ -terminal sequence analysis. 1.2nmole of reduced and alkylated TGF-β₁ was digested in 0.75M urea, 50 mMNH₄ HCO₃. 5 mM dithiothreitol for 24 hours with 1 percent clostripain¹⁵.An additional 1 percent of clostripain was added after 12 hours reactiontime. The reaction products were separated on a Synchropak RPP C18reverse phase column (4.6×250 mm) with a 0-70 percent acetonitrilegradient in 0.1 percent trifluoroacetic acid. Sequence determinationtook place using either an extensively modified Beckman 890C spinningcup sequencer⁶¹ or a vapor phase sequencer as described by Hewick etal.⁶² (Applied Biosystems, model 470A), with amino acid derivativeidentification by reversed phase HPLC on a Rainin Microsorb C-8 column.The amino acid sequence of several peptides was determined. One of thesefragments was the NH₂ -terminal segment, while another large peptideyielded a 37 amino acid sequence which overlapped the NH₂ -terminalsequence and established 60 residues of contiguous sequence.

Unmodified TGF-β₁ was also treated with CNBr. Cleavage at the methionineresidue resulted in the complete loss of biological activity,documenting that at least part of this C-terminal octapeptide is neededfor biological activity (data now shown).

EXAMPLE 2 Isolation of a TGF-β Exon

The approach we followed for the initial identification of a nucleotidesequence encoding TGF-β₁ adopted the "long probe" strategy usedpreviously for TGF-α⁷. Long oligonucleotides designed on the basis ofthe partial protein sequence were used as hybridization probes for theidentification of a TGF-β₁ exon in a human genomic DNA library. TheTGF-β₁ exon was then used as a probe for the isolation of TGF-β₁ cDNAs.

Two 44-base-long deoxyoligonucleotides, βLP1 and βLP2, complementary tosequences coding for amino acids 3 to 17 and 30 to 44, respectively,were chemically synthesized ⁶³,64. The choice of nucleotide sequence wasbased upon the codon bias observed in human mRNAs²⁶. CpG dinucleotides,which are relatively rare in vertebrate DNA²⁷, were avoided wheneverpossible. In addition, sixteen 14-mers were synthesized which arecomplementary to all possible codons for amino acids 13 to 17. Thesedeoxyoligonucleotides and the corresponding amino acid sequence areshown below. ##STR1##

The nucleotides marked with a dot are bases for which there is noambiguity in the codon.

A human genomic DNA library²⁸ was screened under low stringencyhybridization conditions using ³² P-labelled βLP-1 as probe.Approximately 7.5×10⁵ recombinant phage from a human genomic fetal liverlibrary²⁸ were hybridized using low stringency conditions⁶⁵ with the ³²P-labelled 44-mer βLP-1 and βLP-2 oligonucleotides using the "dot blot"analysis method⁶⁷ and Southern hybridization with both oligonucleotideswere digested and probed with the pool of ³² P-labelled 14-mers again bySouthern hybridization. 14-mer hybridizations were performed at 37° C.in 6×SSC, 0.5 percent NP40, 6 mM EDTA, 1X Denhardt's solution and 50μg/ml salmon sperm DNA. Several washes were performed at roomtemperature in 6×SSC before autoradiography. DNA from phage βλ58hybridized with the oligonucleotides βLP-1, βLP-2 and with the 14-merpool. The sequences hybridizing to βLP-2 and the 14-mers were localizedwithin the same 4.2 kbp BamHI fragment, while probe βLP-1 hybridized toa 20 kbp BamHI fragment. The hybridizing BamHI fragments were subclonedinto pBR322. The nucleotide sequence of smaller hybridizing fragmentswas determined by dideoxynucleotide chain termination method⁶⁹ aftersubcloning into M13 derivatives⁷⁰.

The screening of the genomic DNA library resulted in the isolation of anexon coding the part of the TGF-β₁ coding sequence starting at matureresidue 10. In order to obtain the entire TGF-β₁ coding sequence, thisexon was used as a probe to screen a λgt10 based cDNA library derivedfrom human term placenta mRNA.

EXAMPLE 3 Isolation of TGF-β cDNAs

Total RNA was extracted⁷¹ from the different cell sources and thepolyadenylated mRNA fraction was isolated by oligo(dT)-cellulosechromatography ⁷². The cDNA was prepared⁷³ by priming with dT₁₂₋₁₈ orthe deoxyoligonucleotide ACACGGGTTCAGGTAC. The double-stranded cDNA wastreated with nuclease S1 (Miles Laboratories) followed by E. coli DNApolymerase I Klenow fragment (Boehringer Mannheim) and subcloned intoEcoRI cleaved λgt10 as described⁷⁴, except that asymmetric EcoRIlinkers⁷⁵ were used, thus avoiding the need for the EcoRI methylasetreatment. The recombinant phage were plated on E. coli C600 Hfl⁷⁴ andreplica plated onto nitrocellulose filters⁶⁶. These were hybridized with³² P-labelled⁷⁶ restriction fragments of the Example 2 exon at 42° C. in50 percent formamide, 5×SSC, 50 mM sodium phosphate pH 6.8, 0.1 percentsodium pyrophosphate, 5×Denhardt's solution, 50 μg/ml salmon sperm DNAand washed in 0.2×SSC, 0.1 percent SDS at the same temperature. Lowstringency hybridization conditions⁶⁵ were used in the case of the ³²P-labelled deoxyoligonucleotides. The nucleotide sequence of the TGF-β₁cDNA restriction fragments was determined by the dideoxyoligonucleotidechain termination method⁶⁹ after subcloning into M13 phagederivatives⁷⁰. The cDNAs obtained are schematically shown in FIG. 1a.λβC1 was isolated from a human placenta cDNA library using the genomicexon (FIG. 3) as probe. The screening of approximately 750,000 oligo-dTprimed placenta cDNA clones resulted in the isolation of one TGF-β cDNA(λβC1) of about 1,050 bp. The previously determined partial TGF-β₁sequence established the reading frame and revealed the sequence codingfor the complete TGF-β₁ polypeptide. This sequence begins with the NH₂-terminal alanine residue and is followed 112 codons later by a stopcodon, only 20 base pairs from the 3' end. The λβC1 EcoRI cDNA insertwas used in turn to screen the A172 glioblastoma cDNA library leading tothe isolation of λβC3.19. Screening of a specifically primed HT1080fibrosarcoma cDNA library with the ³² P-labelled KpnI-KpnI and theupstream EcoRI-KpnI fragment of the λβC3.19 cDNA insert yielded λβC4.10,4.33 and 4.37. Another similar library was screened with the λβC4.33insert and a synthetic 40-mer corresponding to nucleotides 1-40, leadingto the isolation of λβC5.7b.

Since none of more than seventy TGF-β cDNAs isolated from differentoligo(dT)-primed cDNA libraries contained more than a few nucleotides of3' untranslated region, the 3' unstranslated sequence was determinedusing cloned genomic DNA. Hybridization analysis showed that the 3' endof the λβC1 cDNA insert was present in the genomic DNA phage βλ58. DNAsequence analysis revealed the presence of an exon coding for thecarboxy terminal part of TGF-β₁, followed by the stop codon and the 3'untranslated end (FIG. 1b). An AATAAA hexanucleotide sequence³² wasencountered 500 bp downstream from the termination codon, thuspermitting an assignment of the putative polyadenylation site. Assumingthis is indeed the polyadenylation signal, the calculated size of TGF-β₁mRNA is in close agreement with the 2.3 to 2.5 kb length determined fromthe Northern hybridization experiments (Example 4). Additional screeningof oligo(dT)-primed placenta and HT1080 cDNA libraries using the genomicDNA probe for the 3' untranslated end did not identify a singlehybridizing cDNA phage.

EXAMPLE 4 Diagnostic Method Using TGF-β cDNA Probes

Polyadenylated RNA was recovered from the hepatoma HEP-G2, Wilms tumorTuWi, glioblastoma A172, bladder carcinoma T24, squamous epidermoidcarcinoma A431, mammary carcinoma MCF-7, nasopharyngeal carcinoma KB,fibrosarcoma HT1080, Burkitt lymphoma B-lymphoblasts Daudi and Raji,T-lymphoblast Molt-4. Peripheral blood lymphocytes (PBLs) were preparedand mitogen-induced with staphylococcal enterotoxin B and phorbolmyristate as described⁵³. RNA was harvested in this case after 24 hours.4 μg of polyadenylated mRNA was electrophoresed into formaldehyde-1.2percent agarose gel²⁹ and blotted onto nitrocellulose filters³⁰. The ³²P-labelled⁷⁶ EcoRI cDNA insert of λβC1 was used as probe under highstringency conditions used above. Comparison with the position of the28S and 18S rRNA on the gel suggests a length of 2.3-2.5 kb for theTGF-β mRNA. In some cases a smaller mRNA species may be present,although partial degradation of the mRNA cannot be excluded.

TGF-β mRNA was detectable in all human tumor cell lines including tumorcells of neuroectodermal origin, such as TuWi (Wilms Tumor) and A172(glioblastoma), and the carcinoma cell lines T24 bladder carcinoma, A431(squamous epidermoid carcinoma), MCF-7 (mammary carcinoma) and KB(nasopharyngeal carcinoma). HT1080, a fibrosarcoma derived cell line,which we had chosen as a source of mRNA for the cDNA cloning, containedrelatively high levels of TGF-β mRNA. TGF-β was not only present in celllines derived from solid tumors of meso-, endo- and ectoblastic origin,but was also detectable in tumor cell lines of hematopoietic origin,e.g. Daudi (Burkitt lymphoma B-lymphoblast), Raji (Burkitt lymphomaB-lymphoblast), and Molt-4 (T-cell leukemia). The presence of TGF-β mRNAis not restricted to tumor cells, since it is clearly detectable inplacenta and peripheral blood lymphocyte (PBL) mRNA. Strikingly, thelevel of TGF-β mRNA is significantly elevated after mitogenicstimulation of PBLs. TGF-β mRNA was not detectable in human liver, yetwas present in the HEP-G2 hepatoma cell line. In all cases, the TGF-βmRNA migrated as a species of an apparent length of 2.3 to 2.5 kbases.In some cases a smaller mRNA species of about 1.8 to 1.9 kb may bepresent, although this could be due to partial degradation of the mRNA.

EXAMPLE 5

The plasmid used for recombinant synthesis of TGF-β₁ was pMBTE6. Thefollowing prophetic method for making this plasmid is preferred over themore complex method actually employed in its construction.

p342E⁷⁹ is digested with EcoRI, blunted with E. coli DNA polymerase I(Klenow fragment) and the four dNTPs, digested with SalI and Fragment 1(containing the Amp⁴ gene of pBR322) recovered.

p342E is simultaneously digested with SalI and HindIII and theHBsAG-encoding fragment is recovered as Fragment 2.

Finally, the SV40 genome is simultaneously digested with HindIII andHincII, and the 596 bp fragment containing the SV40 origin and earlypromoter recovered as Fragment 3.

Fragments 1, 2 and 3 are ligated in a three way ligation and theligation mixture is transformed into E. coli strain 294 (ATCC 31446).The transformed culture is plated on ampicillin media plates andresistant colonies are selected. p342E-blunt was recovered from atransformant colony.

p342E blunt is digested simultaneously with HindIII and EcoRI and thelarge vector fragment recovered. This fragment is ligated to apolylinker having the following sequence ##STR2## and the ligationmixture was used to transform E. coli ATCC 31446 as described above.pCVSV-HBs is recovered from an ampicillin-resistant transformant.

pCVSV-HBs is digested with HindIII and EcoRI simultaneously and thevector fragment isolated (the 18 bp HindIII-EcoRI fragment will notappear in the gel due to its small size).

pgD-DHFR-Trunc (European Patent Application 84.305909.8), a plasmidcontaining DNA encoding the herpes simplex gD protein, is simultaneouslydigested with StuI and HindIII and the approximately 760 bp fragmentrecovered which contains DNA encoding the herpes simplex signal peptideand the coding region for the N-terminal part of the mature HSV-1gDprotein. Plasmid pJ2.9 from European Patent Application 84.305909.8 canbe used in the same fashion.

pβC1 (FIG. 1a) is digested with SmaI and BamHI, and the 480 bp fragmentrecovered. This fragment contains most of the sequence coding forpreTGF-β₁ including the sequence coding for the N-terminus of matureTGF-β₁ through residue 314.

pβC1 is digested with BamHI and EcoRI and the 270 bp fragment recovered.These two separate digestions of pβC1 aliquots were conducted becausethe BamHI-EcoRI pβC1 fragment contains a SmaI site. The 270 bp fragmentcontains the sequence coding for the rest of the TGF-β₁ molecule andextends 20 bp beyond the stop codon.

The pCVSV-HBs vector fragment is ligated in a four way ligation with theforegoing 760, 270 and 480 bp fragments. The resulting construction(pCVSVgD) thus contained a hybrid coding sequence (Herpes simplex gD-1signal peptide and part of the gD-1 envelope protein linked in frame tothe preTGF-β₁ precursor fragment) under the control of the SV40 earlypromoter. This hybrid coding sequence is in turn followed by the 3'untranslated sequence and the polyadenylation signal of the hepatitissurface antigen.

pCVSVgD is digested with EcoRI, blunted with Klenow and the four dNTPs,and thereafter digested with PstI. Two fragments are so obtained, withthe fragment including the hybrid coding sequence and the SV40 promoter(fragment A) being recovered.

pCVSVgD is digested with BamHI, blunted with Klenow and the four dNTPs,and thereafter digested with PstI. Four fragments are obtained afterthese digestions. The fragment containing the pBR322 origin and Amp^(r)gene (about 1900 bp) is recovered as Fragment B.

Fragments A and B are ligated and the ligation mixture used to transformE. coli ATCC 31,446. Plasmid pMBTE6 is recovered from an Amp^(r) colony.

Plasmid pMBTE6 was transfected into DHFR deficient CHO cells⁵⁵ togetherwith plasmid pFD11⁹⁰. The latter plasmid encodes DHFR, therebyconferring methotrexate resistance on the transfected cells and allowingfor selection of TGF-β expressing transformants. Any DHFR⁻ mammalianhost cell is suitable for use. Alternatively, one can use any mammalianhost cell, cotransform the host cell with a plasmid encoding neomycinresistance, and identify transformants by their ability to grow inneomycin-containing medium.

The transfected CHO cells were selected by culturing in HGT⁻ medium. Thecells were allowed to grow to confluency in 15 cm diameter plates. Thecells thereafter were cultured in serum-free medium for 48 hours priorto harvest. The culture medium was decanted from the plates and assayedin a soft agar assay for the presence of TGF-β as described⁷⁸.

50 ml supernatant medium was lyophilized and dissolved in 700 μl 4mMHCl-0.1 percent bovine serum albumin. 200 μl of this solution and ofserial three-fold dilutions were assayed. The number of colonies in softagar with a diameter of >82 μm were counted. The maximal response(plateau-value) obtained in the presence of saturating levels of TGF-β₁was about 1500 per plate. Less than 50 colonies were obtained in theabsence of TGF-β₁. A half maximal response was obtained with a 9-folddilution of the sample derived from the cells transformed with anegative control plasmid. Calculations from the values obtained byserial dilution of the MBTE6 supernatant showed that the half maximalvalue was obtained at a 70-fold dilution.

The assays of serial dilutions show that cells transformed with MBTE6and pFD11 synthesize about 8-10 times more TGF-β₁ per ml of medium thando CHO cells transfected with pFD11 alone or with pFD11 together with acontrol plasmid (one which was similar to pMBTE6 except that the Herpescoding sequence is replaced by the sequence coding for the bacterialSTII signal peptide), even prior to subcloning and selection inMTX-containing media. This addition amount of TGF-β is human TGF-β₁.

Since biologically active TGF-β₁ is found in the culture medium it isconcluded that CHO cells cleave preTGF-β in the same fashion as do humancells in vivo to secrete mature native TGF-β. This conclusion is verymuch strengthened by the fact that the slope of the TGF-β₁ concentrationdilution curve in the soft agar is identical for both the endogenousnatural TGF-β₁ and the recombinant TGF-β₁, thus reflecting a similar ifnot identical affinity for the TGF-β receptor.

EXAMPLE 6 Isolation of DNA Encoding TGF-β3

1.5×10⁶ plaques from porcine ovarian cDNAλ library was screened underlow hybridizing conditions with the ³² P-labelled E. coli insert of λβC₁(1050 bp) in pH6.8 hybridization buffer containing 5×SSC, 20% formamide,5×Denhardts, 0.1% Na-pyrophosphate, 0.05M NaPO₄, 0.1% SDS and 50 μg/mlsalmon sperm DNA at 42° C. overnight. Washes were in 2×SSC at 37° C.Phage was purified from positively hybridizing plaques and their DNAinserts were sequenced. The approximately 200 positively hybridizingcDNA inserts fell into three classes: Porcine TGF-β₁ (2 plaques), G-Crich cDNAs which did not encode a TGF-β polypeptide (6) and λ11.3, agene fragment which contained DNA encoding porcine TGF-β₃ downstreamfrom mature residue 10.

The labelled EcoRI insert of λ11.3 was used under high stringencyconditions (as above but 50% formamide, and with washes using 0.1×SSC at42° C.) to rescreen 1×10⁶ plaques from a porcine ovarian cDNA λ library.Of 20 positively hybridizing plaques, one (λ10) contained the entireporcine TGF-β₃ sequence. The combined nucleotide and imputed amino acidsequences encoded by λ10+11.3 are shown in FIGS. 4a-4c.

1×10⁶ plaques from a human ovarian cDNA library were screened withlabelled porcine cDNA. One positive plaque (λhu4) was identified. λhu4has the nucleotide and imputed amino acid sequence set forth in FIGS.4a-4c. FIG. 5 is a comparison of the amino acid sequences imputed fromthe porcine and human TGF-β₃ cDNAs. The candidate start codons for theporcine precursors are boxed.

TGF-β₃ is expressed in recombinant cell culture and recovered therefromin substantially the same way as TGF-β₁, making allowances fordepartures in nucleotide and amino acid sequence as will be apparent tothose skilled in the art. Since the complete precursor for human TGF-β₃is not disclosed, in order to express human TGF-β₃ it will be desirableto reprobe genomic or cDNA libraries for DNA encoding the remainingN-terminal precursor sequence, or ligate DNA encoding the availablehuman sequence (starting at the codon for residue 3) with the DNAencoding the porcine TGF-β₃ precursor through residue 297 (numbered asshown in (FIG. 5), or prepare DNA encoding a heterologous mammalian orviral signal fusion with DNA encoding mature human TGF-β₃.

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We claim:
 1. Isolated nucleic acid encoding TGF-β.
 2. The nucleic acidof claim 1 wherein the nucleic acid is DNA encoding TGF-β that is freeof one or more introns present in genomic DNA.
 3. The DNA of claim 2which is labelled with a detectable moiety selected from the groupconsisting of a fluorescent label, a radioactive atom, and achemiluminescent label.
 4. The DNA of claim 1 which is labelled with adetectable moiety selected from the group consisting of a fluorescentlabel, a radioactive atom, and a chemiluminescent label.
 5. The nucleicacid of claim 1 encoding TGF-β3.
 6. The nucleic acid of claim 5 that islabeled with a detectable moiety selected from the group consisting of afluorescent label, a radioactive atom, and a chemiluminescent label. 7.The nucleic acid of claim 1 wherein the nucleic acid is DNA encodingheterodimeric TGF-β.
 8. The nucleic acid of claim 7 wherein theheterodimeric TGF-β is a heterodimer of TGF-β1 and TGF-β3.
 9. Thenucleic acid of claim 1 encoding TGF-β1.
 10. The nucleic acid of claim 9wherein the nucleic acid is DNA encoding TGF-β3 that is free of one ormore introns present in genomic DNA.
 11. The nucleic acid of claim 10that is labeled with a detectable moiety selected from the groupconsisting of a fluorescent label, a radioactive atom, and achemiluminescent label.
 12. A replicable vector comprising the nucleicacid of claim
 9. 13. A host cell containing the vector of claim
 12. 14.The nucleic acid of claim 9 which is labelled with a detectable moietyselected from the group consisting of a fluorescent label, a radioactiveatom, and a chemiluminescent label.
 15. The nucleic acid of claim 9which is DNA.
 16. The nucleic acid of claim 9 that is cDNA of genomicDNA.
 17. A replicable vector comprising the nucleic acid of claim
 1. 18.The vector of claim 17 wherein the nucleic acid is free of introns. 19.A host cell containing the vector of claim
 18. 20. A host cellcontaining the vector of claim
 17. 21. The cell of claim 20 which is aeukaryotic cell.
 22. The cell of claim 20 which is a bacterial cell. 23.The replicable vector of claim 17 wherein the nucleic acid encodesTGF-β3.
 24. A host cell containing the vector of claim
 23. 25. Thereplicable vector of claim 23 wherein the nucleic acid is DNA free ofintrons.
 26. A host cell containing the vector of claim 25.